(12) Patent Application Publication (10) Pub. No.: US 2007/0122492 A1 Behr Et Al
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US 2007.0122492A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0122492 A1 Behr et al. (43) Pub. Date: May 31, 2007 (54) PLANT EXTRACTS AND Publication Classification DERMATOLOGICAL USES THEREOF (51) Int. Cl. A6IR 36/45 (2006.01) A6IR 36/53 (2006.01) A 6LX 36/899 (2006.01) (76) Inventors: Stephen Behr, Outremont (CA); A6IR 36/8962 (2006.01) Philippe Duret, Quebec (CA); Nathalie A6IR 36/906 (2006.01) Gendron, Neuville (CA); Johane A6IR 36/48 (2006.01) Guay, St-Augustin de Desmaures (CA); A6IR 36/8 (2006.01) Bernard Lavallee, Ste. Foy (CA); A6IR 36/3 (2006.01) Brigitte Page, Quebec (CA) A6IR 36/23 (2006.01) (52) U.S. Cl. ......................... 424/725; 424/750; 424/754; 424/732; 424/760; 424/770; 424/757; 424/756; 424/745 Correspondence Address: HELLEREHRMAN LLP (57) ABSTRACT 275 MODLEFIELD ROAD MENLO PARK, CA 94025-3506 (US) The present invention provides for plant extracts and der matological formulations comprising one or more plant extracts that are capable of inhibiting one or more extracel lular proteases selected from the group of matrix metallo protease-1 (MMP-1), matrix metalloprotease-2 (MMP-2), (21) Appl. No.: 10/533,025 matrix metalloprotease-3 (MMP-3), matrix metallopro tease-9 (MMP-9) and human leukocyte elastase (HLE). The present invention further provides for a rapid method for screening plant extracts to identify those having the above (22) PCT Fed: Nov. 18, 2004 activity that are suitable for incorporation into the derma tological formulations of the invention. The invention also provides for the use of the plant extracts as dermatological agents Suitable for the treatment or prevention of various (86) PCT No.: dermatological conditions, including wrinkling or sagging of the skin, irradiation induced skin and/or hair damage, S 371(c)(1), deepening of skin lines, elastotic changes in the skin, as well (2), (4) Date: Nov. 20, 2006 as for the routine care of the skin, hair and/or nails. Patent Application Publication May 31, 2007 Sheet 1 of 9 US 2007/O122492 A1 FIGURE 1 Potential Plant Optional Pre-Harvest Treatment Harvest Solid Sl Optional Storage Treatment us an X is 88 as a Contact Solid S1 with Solvent A Extraction Process I Separate Extract A m from Solid Matter S2 Potential Extract A Extraction Process II Separate Extract B from Solid Matter S3 Contact Solid S3 with Solvent C Extraction Process III Separate Extract C from Solid Matter S4 Solid S4 Patent Application Publication May 31, 2007 Sheet 2 of 9 US 2007/0122492 A1 FIGURE 2 Potential Extract Optional Separation Procedure(s) Potential Extract Test Aliquots against OC O. O. Extracellular Proteases Does Extract demonstrate inhibitory Potential Extract is an Inconclusive Extract of the activity against one or more extracellular Invention proteases? Potential Extract is not an Extract of the Invention Plant remains a Potential Plant Entire Procedure may be repeated on Potential Plant under varied conditions Patent Application Publication May 31, 2007 Sheet 3 of 9 US 2007/0122492 A1 FIGURE 3 One or More Plants of the Invention Optional Pre-Harvest Treatment Harvest Solid Sl Optional Storage Treatment Extraction Process I, II or II. Process may be sequentially repeated Purification Procedure(s) Quality Control Extract Demonstrating Inhibitory Activity Against One or More Extracellular Proteases Prepare Formulation for Use Patent Application Publication May 31, 2007 Sheet 4 of 9 US 2007/0122492 A1 FIGURE 4 Patent Application Publication May 31, 2007 Sheet 5 of 9 US 2007/O122492 A1 FIGURE 5 Patent Application Publication May 31, 2007 Sheet 6 of 9 US 2007/O122492 A1 FIGURE 6 Dried powdered material Analytical Scale: 2 - 10 g Large scale: 2-3 kg Analytical scale: procedure 1 Large scale: procedure 2 Centrifugation EtOHIMeoH extract EtOHIwater extract Concentration under reduce pressure EtOHIMeOH extract Dilution with 10-5% water Partition with hexane Extract B Hexane extract C 1) Concentration Procedure 2) Solubilization EtOH water 20/80 viv EtOHIMeOh:8515 why 3) Partition with Ethyl acetate 2x100ml protocola) SOHNWater 85/15 wiv 2x100ml (protocol b) Blender (2 x 10 min) Room temperature Procedure 2: Evaporation to dryness OHIMech:85/15 viv (protocola) EtOHWatar: 85/15 yN Aqueous extract E ACOEt extract F maceration(protocol b) percolation A. Room terrperattire Patent Application Publication May 31, 2007 Sheet 7 of 9 US 2007/0122492 A1 — Melilotus alba -e-keratinocytes -O-Fibroblasts 80 6 O -e-keratinocytes -O- Fibroblasts FIGURE 7 Patent Application Publication May 31, 2007 Sheet 8 of 9 US 2007/0122492 A1 1009wÐ (OS:OS)OZH:ng»Dow (08:0Z)OZH:ngXpow (0:001)ozH:ng»Dow FIGURE 8 Patent Application Publication May 31, 2007 Sheet 9 of 9 US 2007/0122492 A1 a 3S. SE. 5E. 5E 5E 5E saE 9 EN S.E on s in o un o o g 2 2 3 ac U nO y ON Ms w wrd R.o N ra a.c -- O cs d c ES o O : E isd C ro cn cs 5 S.E <: S5 s Cd o ?u Vs t cyL c) - Q C > d- ? s - O o 3 U O U g s s s o . --a S.d — 3 is E is FIGURE 9 US 2007/01 22492 A1 May 31, 2007 PLANT EXTRACTS AND DERMATOLOGICAL activity of MMP-1, -2, -3 and -9 in the skin has also been USES THEREOF shown to occur in response to extrinsic factors such as UV exposure (see U.S. Pat. No. 5,837.224). The ageing process FIELD OF INVENTION (both chronological and photo-induced) involves the increased breakdown various components of the ECM in the 0001. The invention pertains to the field of dermatology, skin, notably collagen, elastin and fibronectin. Enhanced specifically within the field of dermatological preparations expression of collagenase (MMP-1) and stromelysin-1 comprising plant extracts. (MMP-3) has been described as playing a central role in connective tissue breakdown in the skin (Brenneisen, et al., BACKGROUND OF THE INVENTION (2002) Ann. N.Y. Acad. Sci., 973:31-43). Similarly, increased expression of serine elastase in hairless mouse 0002 The skin is the most environmentally-stressed models of chronological and photo-ageing was shown to organ in mammals, particularly in humans. Not only is the skin Subjected to germs, toxic chemicals and hostile envi result in increased fibronectin degradation (Labat-Robert, et ronments, it is the only organ directly exposed to ultraviolet al., (2000) J Photochem. Photobiol. B., 57:113-118). light (UV). In addition, the vitality of this organ is a 0005 Elastic fibers are essential extracellular matrix consequence of genetic processes, which over time, lead to macromolecules comprising an elastin core surrounded by a a decrease in the functionality of the skin. Hence, a variety mantle of fibrillin-rich microfibrils. These fibers endow of dermatological conditions may occur as a result of connective tissues such as blood vessels, lungs and skin with ongoing intrinsic factors (for example, chronological age the critical properties of elasticity and resilience (see review ing, disease and allergies) and/or exposure to a number of of elastic fibers by Kielty C M et al: J Cell Sci (2002) extrinsic factors (such as infection, trauma, radiation, toxins 115:2817-2828). Exposure to the sun is known to cause and steroid use). Skin is a highly organized structure con disorganization of elastin in the skin known as "elastosis.” sisting of two principal parts. The outer thinner part, the which is also a hallmark of skin-ageing. Neutrophil elastase epidermis or cuticle, is organised into four or five cell layers has been implicated in elastosis, for example, when com depending on its location. These layers are the stratum pared to normal mice, mice that are deficient in neutrophil corneum, stratum lucidem (usually only present where the elastase are unaffected by exposure to UVB. In addition, an skin is thickened), stratum granulosum, stratum spinosum increase in elastase activity has been observed in the skin and stratum basale. The inner, thicker part of the skin, the following chronic UVB irradiation (Tsukahara Ketal Biol dermis or true skin, is composed of a papillary layer above Pharm Bull 2001:24(9):998-1003). Both a synthetic inhibi and a reticular layer below. The dermis also comprises blood tor of fibroblast elastase and an extract of Sanguisorba vessels, nerves, hair follicles and Sweat glands. The layer officinalis L. inhibited wrinkle formation and maintained below the dermis, the hypodermis, comprises mainly loose skin elasticity in the rat (Tsukahara Ketal Biol Pharm Bull connective tissue and adipose cells and may be considered 2001:24(9):998-1003). part of the skin in that it functions to anchor the epidermis/ 0006 MMPs also play a role in the loss of elastic fibers dermis to the underlying bone and muscle. The hypodermis in skin. Tissue loss during ageing and age-dependent also supplies the dermis with blood vessels and nerves. pathologies are the result of a disturbed regulation of pro 0003. The cells of the skin, like many tissues, are gen teolytic activities in which elastase-type endopeptidases, erally in contact with a network of large extracellular especially MMP-2 and -9, are overactivated (Isnard Netal: macromolecules that occupy the spaces in a tissue between Biomed Pharmacother. 2002 July:56(5):258-64). In addi the component cells and between adjacent tissues. This tion, gelatinase B (MMP-9) has been shown to degrade extracellular matrix (ECM) functions as a scaffolding on fibrillin in human skin tissue sections (Berton Aetal, Matrix which the cells and tissue are supported and is involved Biol 2000:19(2): 139-148). actively in regulating interaction of the cells that contact it.